Sc 398171

Protein samples were separated by SDS–PAGE (10%), blotted onto nitrocellulose membranes and analyzed by chemiluminescence-based immunodetection according to standard procedures. The following primary antibodies were used for incubation at 4°C for 24 h: ms-anti-NFAT5 1:500 (sc-398171, Santa Cruz), rb-anti-histone H3 1:1000 (ab1791, Abcam), rb-anti-alpha-Tubulin 1:1000 (2144, Cell Signaling), ms-anti-DDK 1:1000 (TA50011-100, Origene), ms-anti-SMMHC 1:1000 (14-6400-80, eBioscience), rb-anti-calponin 1:2000 (ab46794, Abcam), or rb-anti-myocardin 1:1000 (sc-33766, Santa Cruz), ms-anti-beta-actin 1:5000 (ab6276, Abcam).

Western blots were performed either with whole protein extracts or with extracts of cytosolic and nuclear proteins on PAGE-SDS gels. For detecting NFAT5, the following Abs were used: Ab3446 (directed against the C-terminal aa1439-1455 of human NFAT5), Ab110995 (directed against an internal region of human NFAT5; both Abcam), sc-398171 (directed against aa67-300 of human NFAT5; Santa Cruz, Biotech) and PAI-023 (directed against a C-terminal peptide of human NFAT5; Affinity BioReagents). NFATc proteins were detected using the 7A6 mAb #556602 (for NFATc1) and the mAb #5062574 (for NFATc2; both BD Pharmingen). As loading control, filters were stained by Ponceau Red for 5 min and/or re-probed with the mAb #ab8227 specific for β-actin. Signals were developed using a chemiluminescence detection system (ThermoFisher Scientific).
For qRT-PCR assays, RNA was isolated from freshly harvested and PBS-washed or from deep-frozen cells using a standard TRIzol/isopropanol protocol. cDNAs were synthesized using the iScript cDNA synthesis kit according to the manufacturer’s instructions (Bio-Rad). Real-time PCR assays were performed using the SYBR green master mix (Applied Biosystems) with the primers presented in Supplementary Table 1.

The extraction of total proteins for cells was carried out with RIPA, and concentrated protein supernatants were measured using a Pierce BCA protein assay kit. Proteins were separated via 4–15% Mini-PROTEAN^® TGX Stain-Free Precast Gel (Bio-Rad, Marnes-la-Coquette, France). The transfer was performed on a nitrocellulose membrane (Bio-Rad) and saturated for over 90 min with 5% milk in tris-buffered saline (TBS) 1X at room temperature. Then, the membrane was incubated overnight at 4 °C with the first antibody against NFAT5 (1:600, sc-398171, Santacruz) or HMGB1 (1:10,000, ab79823, Abcam) diluted in 5% milk in TBS 1X 0.1% tween-20. The membrane was washed with TBS 1X 0.1% tween-20 and then incubated for 90 min at RT with peroxidase-conjugated secondary antibody anti-mouse or anti-rabbit, respectively (1/10,000). After washing, the revelation was completed using an ECL clarity kit (Clarity Western ECL Substrate, BIO-RAD, Marnes-la-Coquette, France) for Western blot and integrated on the ChemiDoc imaging system (Bio-Rad). The relative intensities of the protein bands were analyzed using Image Lab 6.1 software (BIO-RAD, Marnes-la-Coquette, France). Total protein normalization (Bio-Rad), a method allowing normalization using the total protein loaded, was used to normalize the Western blot results.